Ion channel currents are the fundamental units of electrical activity in most organisms. In our nervous system, K+ and Ca2+ channels are critical, and their regulation provides a way to directly control neuronal excitability and release of neurotransmitter (NT) at synapses. The modulations are crucial to basic nervous function and their understanding should contribute to novel modes of medical interventions for a range of disorders involving the brain, nerves and muscles. We focus primarily on the family of KCNQ (Kv7/M-type) K+ channels that underlie several neuronal K+ currents, and also on CaV2.2 (N-type) Ca2+ channels. In particular, we seek to elucidate the molecular mechanisms of Gq/11- mediated pathways that act on these ion channels, and the functional effect of these pathways on release of NT. Both KCNQ and CaV2.2 channels have emerged as being regulated by PIP2, and this project studies the molecular mechanisms of the PIP2-mediated regulation. We use heterologous systems in which the channels, receptors and signaling molecules are expressed in mammalian tissue- culture cells or oocytes, preparations of rodent sympathetic superior cervical ganglion (SCG) neurons, hippocampal neurons grown on astrocyte micro-islands and a co-culture of SCG neurons and mouse cardiomyocytes. In specific aim #1, we will study the biochemical and molecular interactions between PIP2, calmodulin and their binding domains on KCNQ channels using inside-out macropatches, surface plasmon resonance spectroscopy and isothermal calorimetry. We hypothesize PIP2 and CaM to act on overlapping domains on the channels, providing for allosteric cross-talk. In specific aim #2, we will probe the mechanism of receptor-mediated stimulation of phosphatidylinositol 4- and 5-kinases that we hypothesize to underlie the receptor specificity in modulation of M channels. We will also probe the underlying mechanism of receptor-specificity in Ca2+i signaling, focusing on the proteins IRBIT, DAG- kinase, and small GTPases of the Rho family. In specific aim #3, we investigate the functional role of M-channel regulation in control over NT release, using two innovative approaches. The first is a co- culture in which SCG neurons make adrenergic synapses on spontaneously-beating cardiomyocytes cultured in a dish, using cells taken from wild-type or receptor knock-out mice. The second uses isolated hippocampal neurons which form autapses, allowing the input/output relation between action potential and NT release to be directly determined. The molecules and signaling pathways that we study have broad relevance to human health and disease, as these channels play a dominant role in regulating excitability of neurons, and their regulation likely underlies changes in emotional state, memory and regulation of body organs. Thus, our research should provide the basis for the development of novel modes of therapeutic intervention for a variety of nervous diseases.